染色质高级结构调控基因活性的机制研究

It is becoming increasingly evident that precise regulation of eukaryotic gene expression requires communication between cis-acting elements through alterations in higher order chromatin organization. During gene activation or silencing, distal regulatory elements, such as enhancers, locus control regions, differentially methylated regions, and insulators, adopt a close proximity to their target genes with the looping out of the intervening DNA. SHC1 contains two tandem promoters producing p52Shc and p66Shc respectively. These two proteins execute opposing functions in mediating anchorage dependent growth. p52Shc, which is generated from upstream promoter, mediates anchorage-related proliferative signals whereas p66Shc (generated from downstream promoter) enforces anoikis. Our recent work demonstrated that in HUVECs which expresses comparable level of p66Shc and p52Shc, the two tandem promoters are mutually exclusively activated in two alleles. This exclusive monoallelic transcription manner might be maintained by the monoallelic long range intrachromasomal interaction between two tandem promoters. The colocalization of two promoters inhibits the activity of upstream promoter and facilitates transcription initiating from downstream promoter. In this proposal, we are going to study the molecular mechanisms by which the colocalization of promoters regulates promoter usage, including how the physical interaction between two promoters inhibits activity of upstream promoters and what epigenetic marks distinguish the two alleles. In addition, we are going to perform genomic wide screen for genes that colocalize with SHC1 in HUVECs and the change of SHC1 neighborhoods in TGF-b2 induced reprogramming. Completion of this project will shed a light in understanding the cause and consequence of chromatin architecture in gene transcription.

真核细胞中源于功能DNA片段相互作用的染色质高级结构与基因活性密切相关，但其如何调控基因活性并不清楚。我们近期研究发现，内皮细胞中含有两个启动子的SHC1其上下游启动子分别在不同的等位基因活跃，而维持这种单等位基因表达的主要因素很可能是等位基因特异性的不同启动子间相互作用，这种染色质高级结构通过未知机制抑制上游启动子活性促进转录由下游启动子起始，这一发现提出了染色质高级结构调控基因转录的新模式，本项目将以胎儿脐静脉分离的HUVEC为主要实验体系，利用3C技术，深入研究这种启动子间相互作用的形成机制及其功能，包括这种启动子间相互作用如何调控启动子活性；何种表观遗传标记指导等位基因特异性染色质环的形成等；同时应用4C结合高通量测序技术研究HUVEC中以SHC1为核心的三维染色质高级结构在细胞重编程过程中的动态变化，研究结果将有助于人们对染色质高级结构形成及功能的理解。

人类基因组中超过60%的基因具有多个启动子，其中很多基因的不同启动子编码的蛋白不同，对细胞的功能起着相反的作用，并且与疾病相关，细胞如何调控不同启动子的活性目前还不清楚。SHC1基因含有两个启动子，分别编码p66和p52，p66促进细胞凋亡，而p52促进细胞增殖，这两个蛋白在内皮细胞中同时表达。利用SHC1为模型，我们发现内皮细胞中p66和p52分别在不同的等位基因表达，这种表达模式能够很好的避免上游启动子的转录对下游启动子产生影响。进一步的机制研究发现，不同启动子之间的相互作用是决定这种表达模式的关键因素，启动子间相互作用仅发生于一个等位基因，这种相互作用将上游启动子置于一个小的环状结构中，不利于RNA聚合酶的结合或者转录的延伸，因此抑制了上游启动子的活性，释放了下游启动子，使得转录由下游启动子进行。 我们的这一发现提出了基因转录调控的新机制，并证实染色质高级结构在调控基因转录中的重要作用。